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Published in final edited form as: Phytochemistry. 2007 Sep 21;69(2):553–557. doi: 10.1016/j.phytochem.2007.08.009

Furoquinoline Alkaloids of Ertela (Monnieria) trifolia (L.) Kuntze from the Suriname Rainforest

Shugeng Cao a, Adnan J Al-Rehaily b, Peggy Brodie a, Jan H Wisse c, Etienne Moniz c, Stan Malone d, David G I Kingston a,*
PMCID: PMC2245804  NIHMSID: NIHMS39531  PMID: 17889047

Abstract

7-(2′-Hydroxy-3′-chloroprenyloxy)-4,8-dimethoxyfuroquinoline (1) and 6-(2′-hydroxy-3′-chloroprenyloxy)-4,7-dimethoxyfuroquinoline (2), together with ten known compounds have been isolated from the aerial parts of Ertela (Monnieria) trifolia (L.) Kuntze. All the isolates were tested for antiproliferative activity against the A2780 human ovarian cancer cell line.

Keywords: Ertela trifolia, Monnieria trifolia, Rutaceae, Furoquinoline alkaloids, Acridone alkaloids, Furofuran lignans, NMR, Cytotoxicity

1. Introduction

In our continuing search for bioactive molecules from the Suriname and Madagascar rainforests as part of an International Cooperative Biodiversity Group (ICBG) program (Cao et al, 2007) we obtained a methanol extract of the aerial parts of Ertela trifolia (L.) Kuntze (Rutaceae). This extract (BGVS 320017) showed antiproliferative activity against the A2780 human ovarian cancer cell line, with an IC50 value of 20 μg/mL. Ertela trifolia is also known as Monnieria trifolia, and it has been the subject of several previous phytochemical studies (Bhattacharyya et al., 1981, 1983, 1984; Bulhoes et al., 1976; Cave et al., 1971; Fouraste et al., 1973; Keita et al., 1985; Moulis et al., 1981; Rouffiac et al., 1969). To the best of our knowledge, none of the Monnieria sp. have been investigated for their bioactivities. In the present communication, we describe the A2780 guided-separation of twelve alkaloids, the structure elucidation of the two new alkaloids 7-(2′-hydroxy-3′-chloroprenyloxy)-4,8-dimethoxyfuroquinoline (1) and 6-(2′-hydroxy-3′-chloroprenyloxy)-4,7-dimethoxyfuroquinoline (2), and the reassignment of the structure of a previously isolated compound as 13.

2. Results and discussion

Compound 1 was obtained as pale yellow powder. The EIMS of 1 showed the presence of a molecular ion peak at m/z 366 [M+1]+ corresponding to the molecular formula C18H20ClNO5, together with a peak at m/z 368 (34% relative to the molecular ion peak) due to 37Cl isotope, which confirmed the presence of a chlorine atom. The UV spectrum exhibited absorptions at λmax 249 nm and a broad band in the region 300-345 nm, typical of a furoquinoline alkaloid. The 1H NMR spectrum of 1 showed signals for two olefinic doublets, two methoxy groups, two methyl groups, one oxygenated methine, one oxygenated methylene, and two aromatic doublets. The 1H and 13C NMR spectral data (Table 1) of 1 were similar to those of 3, except for the C5 side chain at C-7. The HMBC correlations from H-5 (δ 7.95, d, J = 9.3 Hz) and 4-OMe (δ 4.43, s) to C-4 (δ 156.7), and 8-OMe (δ 3.94, s) to C-8 (δ 141.8), together with the ROESY correlations between H-2 (δ 8.00, d, J = 2.8 Hz) and H-3 (δ 7.45, d, J = 2.8 Hz), H-3 and 4-OMe, H-5 and H-6 (δ 7.44, d, J = 9.3 Hz), and H-6 and H2-1′ (δ 4.49, dd, J = 2.8, 10.2 Hz; 4.14, dd, J = 7.5, 10.2 Hz) demonstrated that 1 was a 4,8-dimethoxyfuroquinoline alkaloid substituted at C-7 (Figure 1). HMBC correlations were observed from 2′-OH (δ 5.77, d, J = 6.1 Hz) to C-1′ (δ 71.3), C-2′ (δ 75.9), and C-3′ (δ 73.0), and from H3-5′(δ 1.64, s) to C-2′, C-3′, and C-4′ (δ 27.9). Therefore, based on the EIMS spectrum of 1, the functional group at C-3′of the side chain at C-7 must be -Cl, and the substituent at C-7 was then established as 2′-hydroxy-3′-chloro-3′-methyl butoxy. Hence, the structure of 1 was determined as shown.

Table 1.

1H and 13C NMR spectral data for compounds 1, 2, and 13 (DMSO-d6)

no. 1Ha
 no. 13Cb
1 2 13 1 2 13
2 8.00 d (2.8) 7.95 d (2.8) 7.90 d (2.6) 2 143.8 143.0 143.6
3 7.45 d (2.8) 7.42 d (2.8) 7.38 d (2.8) 3 105.4 105.2 105.9
3a 3a 114.2 101.8 102.5
4 4 156.7 155.0 155.7
4a 4a 101.8 112.0 112.8
5 7.95 d (9.3) 7.49 s 7.44 s 5 117.7 101.1 101.4
6 7.44 d (9.3) 6 113.8 146.6 147.7
7 7 151.4 152.4 153.1
8 7.29 s 7.26 s 8 141.8 106.6 107.0
8a 8a 140.8 141.8 142.3
9a 9a 163.8 162.5 163.1
4-OMe 4.43 s 4.43 s 4.40 s 4-OMe 59.2 59.2 59.8
7-OMe 3.93 s 3.90 s 7-OMe 55.5 56.2
8-OMe 3.94 s 8-OMe 60.8
1′ 4.49 dd (2.8, 10.2)
4.14 dd (7.5, 10.2)		 4.40 dd (2.2, 10.2)
4.06 dd (7.5, 10.2)		 4.30 dd (2.2, 10.2)
4.06 dd (7.5, 10.2)		 1′ 71.3 70.2 71.6
2′ 3.91 m 3.92 m 3.92 m 2′ 75.9 75.4 76.3
3′ 3′ 73.0 73.0 71.0
4′ 1.59 s 1.60 s 1.12 s 4′ 27.9 27.8 24.8
5′ 1.64 s 1.64 s 1.16 s 5′ 29.6 29.5 27.9
2′-OH 5.77 d (6.1) 5.78 d (6.3) 5.16 d (5.5) 2′-OH
3′-OH 4.59 s
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a

δ (ppm) 500 MHz; multiplicities; J values (Hz) in parentheses.

b

δ (ppm) 125 MHz.

Figure 1.

Figure 1

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Key HMBC and ROESY correlations for 1

The EIMS of 2 also showed a molecular ion peak at m/z 366 [M+1]+ corresponding to the molecular formula C18H20ClNO5, together with a peak at m/z 368 (36% relative to the molecular ion peak) due to 37Cl isotope, which demonstrated that 2 and 1 were isomers. The 1H NMR spectrum (Table 1) of 2 exhibited peaks for two olefinic doublets, two methoxy groups, two methyl groups, one oxygenated methine, one oxygenated methylene, and two aromatic singlets. In the HMBC spectrum, 3J correlations from 4-OMe (δ 4.43, s) to C-4 (δ 155.0), 7-OMe (δ 3.93, s) to C-7 (δ 152.4), and H-5 (δ 7.49, s) to C-4 were observed, and in the ROESY spectrum, correlations between H-2 (δ 7.95, d, J = 2.8 Hz) and 4-OMe to H-3 (δ 7.42, d, J = 2.8 Hz), and 7-OMe and H-8 (δ 7.29, s) were exhibited. The ROESY correlation between H-5 and H2-1′ (δ 4.40, dd, J = 2.2, 10.2 Hz; 4.06, dd, J = 7.5, 10.2 Hz) indicated that 2′-hydroxy-3′-chloro-3′-methyl butoxy was located at C-6. Hence, the structure of 2 was determined as 6-(2′-hydroxy-3′-chloroprenyloxy)-4,7-dimethoxyfuroquinoline.

Neither compound 1 nor 2 showed any optical rotation, suggesting that both were racemic. This observation, coupled with the fact that the isolation of chlorine-containing plant products is unusual, made it important to demonstrate that compounds 1 and 2 were not artefacts caused by ring opening of an epoxide with HCl. The crude extract was thus examined by LC-MS. A peak with the same retention time and molecular ion as compounds 1 and 2 was detectable in this crude extract, demonstrating that these compounds were present in the crude extract and were not an artifact of isolation. Since the extract was prepared by simple room temperature extraction of the plant material with methanol, and had never been treated with HCl, it is unlikely that hydrochlorination could have occurred at this stage, and the compounds are thus presumably genuine natural products. Although chlorinated prenyl groups are rare, they are not unknown. As one example Toddalia asiatica, also a member of the Rutaceae family, yielded the chlorinated prenyl derivative 5,7-dimethoxy-6-(3′-chloro-2′-hydroxy-3′-methylbutyl)coumarin (Sharma et al., 1981).

The structure of 6-(2′-hydroxy-3′-chloroprenyloxy)-4,7-dimethoxyfuroquinoline (2) was previously published under the name chlorodesnkolbisine (compound AR5, Al-Rehaily et al., 2003). A comparison of the NMR data of 2 with those of AR5 indicated that the two compounds were different. A sample of AR5 was then supplied by Dr. Al-Rehaily and investigated by MS and NMR spectroscopy. These data indicated that it was in fact nkolbisine (13) (Ayafor et al., 1982).

The structures of the ten known compounds isolated in this study were determined to be chaplophytin-B (3) (Ali et al., 2001), 7-(2′-hydroxy-3′-methoxy-prenyloxy)-4,8-dimethoxyfuroquinoline (4) (Akhmedzhanova et al., 1975), 7-isopentenyloxy-γ-fagarine (5) (Dreyer, 1969), skimmianine (6) (Chakravarty et al., 1999), 4,8-dimethoxyfuro[2,3-b]quinoline (7) (Ito et al., 2004), tecleanatalesine B (8) (Tarus et al., 2005), methylnkolbisine (9) (Al-Rehaily et al., 2003), 1-hydroxy-2,3-dimethoxy-10-methylacridin-9(10H)-one(10) (Bergenthal et al., 1979), sesamin (11) (Hsieh et al., 2005), and fargesin (12) (Brown et al., 2001) by comparison of their spectroscopic data with literature data.

All the isolates were tested against A2780 human ovarian cancer cell line. Compounds 1, 2, 5, 6, 8, 9, and 10 showed weak antiproliferative activity with IC50 values of 13, 9, 13, 13, 16, 14, 12 μg/mL, respectively.

3. Experimental

3.1. General

Optical rotations were recorded on a Perkin-Elmer 241 polarimeter. IR and UV spectra were measured on MIDAC M-series FTIR and Shimadzu UV-1201 spectrophotometers, respectively. NMR spectra were obtained on a JEOL Eclipse 500 and an Inova 400 spectrometer. The chemical shifts are given in δ (ppm), and constants are reported in Hz. Mass spectra were obtained on a JEOL JMS-HX-110 instrument, in the positive ion mode. HPLC was performed on a Shimadzu LC-10AT instrument with a semi-preparative phenyl Varian Dynamax column (5 μm, 250 × 10 mm) and a preparative C18Varian Dynamax column (8 μm, 250 × 21.4 mm). Finnigan LTQ LC/MS was also used for EIMS analysis of compounds 1, 2, and 13.

3.2. Plant material

A collection of stems, twigs, leaves, and flowers of Ertela trifolia (L.) Kuntze (also known as Monnieria trifolia) was made in late 2000 at Solan; its local name is: Kofimaka. Voucher specimens have been deposited at the National Herbarium of the University of Suriname, Paramaribo, Suriname.

3.3. Extraction and isolation

The plant samples were dried, ground, and extracted with MeOH to give extracts BGVS 320017. Extract BGVS 320017 (1.9 g, IC50 20 μg/mL) was suspended in aqueous MeOH (MeOH-H2O, 9:1, 100 mL) and extracted with hexanes (3 × 100 mL portions). The aqueous layer was then diluted to 70% MeOH with H2O and extracted with CH2Cl2 (3 × 100 mL portions). The CH2Cl2 extract (896 mg) was active with an IC50 of 15 μg/mL, while both the hexane and aqueous MeOH extracts were inactive. The CH2Cl2 extract was chromatographed on an open C18 column (130 × 22 mm) using H2O-MeOH-CH2Cl2 (80:20:0 to 0:100:0, then 0:80:20) to yield three fractions, I [80 mg, inactive], II [700 mg, IC50 12 μg/mL], and III [100 mg, inactive]. Fraction II furnished 12 subfractions (A-L) after HPLC separation on a C18 column (60% MeOH/H2O in 30 min, then to 100% MeOH/ H2O in 10 min, 10 mL/min). Further separation of subfractions B-F and H-K was carried out by preparative Si gel TLC to afford 12 compounds. Subfraction B yielded compound 3 [1.2 mg, CHCl3-MeOH (100:2), Rf0.2]; subfraction C, 7 [1.2 mg, CHCl3-MeOH (100:2), Rf0.4] and 12 [0.6 mg, CHCl3-MeOH (100:2), Rf0.3]; subfraction D, 6 [0.6 mg, CHCl3-MeOH (100:2), Rf0.3]; subfraction E, 9 [0.8 mg, CHCl3-MeOH (100:2), Rf0.5] and 10 [1.1 mg, CHCl3-MeOH (100:2), Rf0.7]; subfraction F, 4 [1.1 mg, CHCl3 (with 0.5% MeOH), Rf0.15]; subfraction H, 2 [0.6 mg, CHCl3-MeOH (100:2), Rf0.5] and 11 [0.6 mg, CHCl3-MeOH (100:2), Rf0.2]; subfraction I, 1 [1 mg, CHCl3-MeOH (100:1), Rf0.3]; and subfractions J and K, 5 [1 mg, CHCl3, Rf0.1] and 8 [1 mg, CHCl3, Rf0.15].

3.3.1. 7-(2′-Hydroxy-3′-chloroprenyloxy)-4,8-dimethoxyfuroquinoline (1)

Light yellow powder. [α]D20 0° (c; 0.10 in MeOH); UV λmax (MeOH) nm (log ε): 249 (4.68), broad band in region 300-345 nm [320 (3.97)]; IR (film) νmax cm-1 2934, 1590, 1474, 1363, 1251, 1084, 1016; 1H NMR (500 MHz, CDCl3): 1.68 (3H, s, H3-4′), 1.69 (3H, s, H3-5′), 3.31 (1H, d, J = 4.0 Hz, 2′-OH), 4.02(1H, m, H-2′), 4.14 (3H, s, 8-OMe), 4.26 (1H, dd, J = 8, 10.2 Hz, H-1′), 4.45 (3H, s, 4-OMe), 4.56 (1H, dd, J = 3.2, 10.2 Hz, H-1′), 7.07 (1H, d, J = 2.8 Hz, H-3), 7.25 (1H, d, J = 9.2 Hz, H-6), 7.61 (1H, d, J = 2.8 Hz, H-2), 8.03 (1H, d, J = 9.2 Hz, H-5); 1H (500 MHz, DMSO-d6): and 13C (125 MHz, DMSO-d6) NMR: see Table 1; (+)-EIMS m/z (rel. int. %): 368 (34), 366 (100); HRFABMS m/z 366.1077 (calcd for C18H21ClNO5, 366.1108).

3.3.2. 6-(2′-Hydroxy-3′-chloroprenyloxy)-4,7-dimethoxyfuroquinoline (2)

Light yellow powder. [α]D20 0° (c; 0.06 in MeOH); UV λmax (MeOH) nm (log ε): 247 (4.57), broad band in region 300-345 nm [320 (4.03)]; IR (film) νmax cm-1: 2935, 1617, 1499, 1375, 1255, 1093; 1H NMR (500 MHz, CDCl3): 1.72 (3H, s, H3-4′), 1.73 (3H, s, H3-5′), 3.00 (1H, d, J = 4.4 Hz, 2′-OH), 4.00(3H, s, 7-OMe), 4.14 (1H, m, H-2′), 4.20 (1H, dd, J = 7.6, 10.2 Hz, H-1′), 4.45(3H, s, 4-OMe), 4.49 (1H, dd, J = 2.4, 10.2 Hz, H-1′), 7.05 (1H, d, J = 2.8 Hz, H-3), 7.34 (1H, s, H-8), 7.57 (1H, s, H-5), 7.58 (1H, d, J = 2.8 Hz, H-2); 1H (500 MHz, DMSO-d6): and 13C (125 MHz, DMSO-d6) NMR: see Table 1; (+)-EIMS m/z (rel. int. %): 368 (36), 366 (100); HRFABMS m/z 366.1077 (calcd for C18H21ClNO5, 366.1108).

3.4. Antiproliferative Bioassays

The A2780 ovarian cancer cell line assay was performed on compounds 1-4 at Virginia Polytechnic Institute and State University as previously described (Cao et al., 2007). The A2780 cell line is a drug - sensitive human ovarian cancer cell line (Louie et al., 1985).

Supplementary Material

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Figure 2.

Figure 2

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Key HMBC and ROESY correlations for 2

Figure 3.

Figure 3

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Acknowledgement

This project was supported by the Fogarty International Center, the National Cancer Institute, the National Science Foundation, the National Heart, Lung and Blood Institute, the National Institute of Mental Health, the Office of Dietary Supplements, and the Office of the Director of NIH, under Cooperative Agreement U01 TW000313 with the International Cooperative Biodiversity Groups, and this support is gratefully acknowledged. We thank Mr. B. Bebout for obtaining the mass spectra and Mr. T. Glass for assistance with the NMR spectra.

Footnotes

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